Detection of Anthrax Detection of Anthrax Spores in MailSpores in Mail

Florine C. ClearyFlorine C. Cleary

20022002

AnthraxAnthrax• Rare disease in humans (1/100,000 in natural environment)• Three types of anthrax in humans:

– cutaneous (infection of skin, eyes and/or mucous membranes)– gastrointestinal (infection of intestinal mucosa)– inhalation (infection of lungs – obtained by inhaling spores)

• Detection of anthrax spores has recently become a Detection of anthrax spores has recently become a concern for us all.concern for us all.

These are the three letters These are the three letters sent containing sent containing AnthraxAnthrax

““This is nextThis is nextTake penacilin nowTake penacilin nowDeath to AmericaDeath to AmericaDeath to IsraelDeath to IsraelAllah is Great”Allah is Great”

““You can not stop usYou can not stop usWe have anthraxWe have anthraxYou die now.You die now.Are you afraid?Are you afraid?Death to AmericaDeath to AmericaDeath to IsraelDeath to IsraelAllah is greatAllah is great””

““This is nextThis is nextTake penacilin nowTake penacilin nowDeath to AmericaDeath to AmericaDeath to IsraelDeath to IsraelAllah is GreatAllah is Great””

Background

• Five peopleFive people dieddied in America last fall as a result in America last fall as a result of handling anthrax-tainted mail.of handling anthrax-tainted mail.

• Two of the people killed were Two of the people killed were postal employeespostal employees who worked at the Brentwood facility, which who worked at the Brentwood facility, which processes mail for the federal government processes mail for the federal government

• ThisThis led to a massive campaign by the U.S. Postal led to a massive campaign by the U.S. Postal Service Service to sanitize mailto sanitize mail, especially that destined , especially that destined for politicians on Capitol Hill.for politicians on Capitol Hill.

Potential Health RisksPotential Health RisksInvolved With IrradiationInvolved With Irradiation

• “Following the ‘clean up’ and new methods implemented by the US postal service, at least 73 Senate employees reported symptoms such as headaches, bloody noses, skin rashes and dry mouth after handling mail that was irradiated to kill any possible anthrax spores. So an inquiry was begun into the possible ill-effects from irradiated mail and the clean-up of the Senate's Hart building.” –Washington Post

• “At least 87 suburban postal workers who handled irradiated mail have reported health problems including nausea, headaches and breathing problems, union leaders say.” -CNN

Further Problems With Irradiated Mail

• Irradiation can have damaging effects on materials (computer media erased and melted, documents destroyed, precious stones ruined, etc.)

• Irradiated mail has been reported to catch fire

Close-up of 1949 Washington quarter dollar showing the bubbling of the plastic encapsulation holder after going through theirradiation process. The coin was “bright white” before irradiation.

AnthraxAnthrax• Bacillus anthracis is found in two forms:

– vegetative cells (metabolizing rod-shaped bacteria, “awake”)

– endospore (non-metabolizing, “dormant” or “hibernating”)

• Bacterial spores are able to withstand extreme treatment, can be aerosolized, and used as a biological weapon.

Pics from the Official US Department of Defense anthrax website (Oct 2001)

Bacillus Anthracis sporesBacillus Anthracis vegetative cells

GOALGOAL Development of methodology and Development of methodology and

instrumentation for the detection of bacterial instrumentation for the detection of bacterial spores in mailspores in mail

Development of a Method for the Detection of Spores Inside Mail

Intrinsic Fluorescence Detection:

– requires NONO reagents (detection relies on optical properties of components found naturally in spores)

– requires NONO sample handling (no opening of mail or direct contact with potentially contaminated mail)

– requires NONO sample processing (no time required for biological ‘grow-out’ or chemical steps)

– can be adapted to existing mail sorting equipment

How Intrinsic FluorescenceDetection Works

envelo p esp o res

exc itatio n light emitted light

• light of a specific energy is directed towards the sample• components of the spores absorb the excitation light and then spontaneously emit light of a different color• the emitted light is detected

Fluorescence Properties of SporesFluorescence Properties of SporesBacterial spores have several intrinsic fluorescent Bacterial spores have several intrinsic fluorescent components that can be used for their detectioncomponents that can be used for their detection..

B. thuringiensis Spores & CellsProtein and DNA Fluorescence

300 350 400 450 5000

5000

10000

15000Cells

Spores

Wavelength

Flu

ore

scen

ce

B. thuringiensis Spores & CellsCalcium Dipicolinate Fluorescence

350 400 450 500 550 6000

50

100

150

200

250

300

350Cells

Spores

Wavelength

Flu

ore

scen

ce

B. thuringiensis Spores & CellsOther Component Fluorescence

700 750 800 850 9000

10

20

30Cells

Spores

Wavlength

Flu

ore

scen

ce

Protein & DNAProtein & DNA Ca-DipicolinateCa-Dipicolinate Other ComponentsOther Components

Fluorescence Properties of Spores Fluorescence Properties of Spores and Vegetative Cellsand Vegetative Cells

Spore GerminationSpore Core Kinetics

0.0 0.1 0.2 0.3 0.4 0.5 0.60

2500

5000

7500

10000Ca-Dipicolinate

Other Component

Time, min.

Pea

k A

rea

Spore GerminationCellular Metabolite Kinetics

0 10 20 30 40 50 600

1000

2000

3000

4000

5000

6000NADH

DPNH

Flavoproteins

Hemoproteins

Time, min.R

elat

ive

Flu

ore

scen

ce

Calcium dipicolinate and other core components are lostquickly when spores germinate. Other fluorophores usedfor microbial detection are inappropriate for spore detection.

Fluorescence Properties of Paper

Paper also exhibits fluorescence when excited with the light used to excite spore components.

The fluorescence from the paper must also be determined to ensure that spores are actually detected.

envelo p esp o res

exc itatio n light

sp o re emis s io n

p ap er emis s io n

Detection of SporesInside Sealed Envelopes

(5 mg sample size)

0

25

50

75

100

EnvelopeOnly

Spores inEnvelope

Flu

ore

scen

ce S

ign

al

The background-corrected signal from spores sealed inside an envelope are shown in the bar graph.

Fluorescence Properties of PaperFluorescence Properties of PaperThe fluorescence properties of 45 kinds of paper and 5 kinds of envelopes were investigated to determine if they would interfere with the detection of spores.

Envelope Fluorescence

650 700 750 800 8500

50

100

150Envelope

Secruity

Manilla

Card Stock

Tyvek

Wavelength

Flu

ore

scen

ce

Paper Fluorescence

650 700 750 800 8500

250

500

750

Wavelength

Flu

ore

scen

ce

The wavelength of light emitted from the other components of spores are shown with the red arrow in the spectra above. Wavelengths used to determine the background of the paper are shown in blue. Both the excitation light and emission light are able to penetrate the envelope making detection of spores inside mail possible.

The wavelengths of light used to determine the background of the paper are shown in blue on the spectra of spores.

Fluorescence of VariousBacterial Spores

650 700 750 800 850 9000

100

200

300

400

500

600B. thuringiensis

B. megaterium

B. subtilis

Wavelength

Fluo

resc

ence

Fluorescence Properties Between Fluorescence Properties Between Spores of Different SpeciesSpores of Different Species

Differences between the fluorescence spectra of bacterial and fungal (e.g., mold) spores can be exploited to differentiate between the species. The method to detect bacterial spores described herein is not “fooled” by mold spores.

Ca-Dipicolinate Other Components

Yeast and Bacterial Spore Solutions(Ca-Dipicolinate Fluorescence)

350 400 450 500 5500

100

200

300

400

500

600Saccharomyces cerevisiae Spores

Bacillus thruingiensis Spores

Wavlength

Flu

ore

scen

ce

Yeast and Bacterial Spore Solutions(Other Component Fluorescence)

700 750 800 850 9000

25

50

75

100

Wavelength

Flu

ore

scen

ce

Hoax Material Interference?

The background-corrected signal from ca. 10 mg of bacterial spores and hoax materials inside a folded piece of paper are shown in the bar graph below.

Detector Signal Output

Sp

ore

s

Ba

by

Po

wd

er

Co

rn S

tarc

h

Je

llo

Po

wd

ere

d M

ilk

Flo

ur

(ble

ac

he

d)

Po

wd

ere

d S

ug

ar

Wh

ite

Co

co

a

Gy

ps

um

-50

0

50

100

150

200

250

300

Sig

nal

Detector Prototype

Further Work

• determine detection limits of method with various spore species

• demonstrate detection with Bacillus anthracis spores

• investigate adapting detector to high-speed mail sorters